Over the last several decades, optical analysis methods have emerged as useful and broadly applicable analytical tools for detecting and characterizing trace components in wide variety of media. In optical analysis methods, electromagnetic radiation is provided to a sample and interacts with components of the sample. The interaction between incident electromagnetic radiation and the sample generates scattered, transmitted and/or emitted electromagnetic radiation that is collected and detected. The intensities, wavelength distribution, polarization states, scattering angles or any combination of these properties of the detected light provides information relating to the composition, concentration, physical state and/or chemical environment of sample components. Optical analysis methods that have been demonstrated as especially useful for characterizing trace components include absorption and emission spectroscopy techniques, Raman and Mie scattering analysis methods, magnetic resonance spectroscopy methods and multidimensional optical spectroscopy techniques
Optical analysis methods provide a number of benefits particularly advantageous for characterizing cellular and noncellular components of biological systems. First, optical analysis methods are applicable to a wide range of biological systems and biological materials, as most biologically significant molecules, such as peptides, oligonucleotides and lipids, and aggregates thereof absorb, scatter and/or affect the polarization states of electromagnetic radiation in ultraviolet, visible and infrared regions. Second, many optical analysis methods provide selective and sensitive means of identifying and characterizing biological materials, particularly methods employing selective optical labeling techniques such as fluorescent labeling or infrared tagging. Third, optical methods often constitute nondestructive characterization methods, thereby allowing components of a biological sample to be analyzed without significantly affecting their biological activities, compositions or physical states. Fourth, optical analysis methods provide in situ, real time detection in static and flowing systems. Finally, recent availability of inexpensive sensitive photodetectors and stable optical sources operating in the ultraviolet, visible and infrared regions of the electromagnetic spectrum makes high throughput analysis using optical methods both commercially and technically practicable.
As a result of these benefits, optical analysis methods are widely used for identifying, classifying and sorting biological materials. Optical flow cytometry, for example, has been demonstrated as useful in a range of diverse experimental settings including immunophenotyping applications, DNA analysis, functional assays, cellular sorting, quantitative analysis of cellular and noncellular particles, and, clinical diagnosis and therapy. In flow cytometry, a suspension comprising cellular and/or noncellular particles of interest are injected into a faster flowing stream of fluid, which provides a sheath around the particles thereby producing laminar flow. The sheath fluid is pumped much faster than the sample in a process known as hydrodynamic focusing, which minimizes clogging and precisely centers sample streams of particles in a small analysis volume. The continuous laminar flow of particles spatially segregates particles such that they pass through an optical detection region where they are characterized. In the optical detection region, particles interact, preferably one at a time, with one or more incident beams of electromagnetic radiation, such as laser light having a selected wavelength distribution, thereby generating scattered, transmitted and/or emitted electromagnetic radiation that is detected as a function of time. Optical measurements typically used in conventional optical flow cytometry systems for characterizing cellular material include low angle forward scattered light intensities for characterizing cell diameter and orthogonal scattered light intensities for determining the quantity of granular structures in a cell.
Fluorescent detection methods are widely employed in convention optical flow cytometry systems, particularly in combination with selective fluorescent labeling techniques, wherein fluorescence intensities corresponding to a plurality of wavelength distributions are simultaneously detected for each particle passing through the optical detection region. Labeled probes and a plethora of fluorescent dyes, stains and intercalators aid in the detection of a wide variety of cell types and cell components. In some methods, fluorescent antibodies are used to measure the densities of specific surface receptors of cellular analytes, and thus to distinguish subpopulations of differentiated cell types. Intracellular components are also routinely detected and quantified using fluorescent probes in combination with optical flow cytometry, including total intracellular DNA, specific nucleotide sequences in DNA or mRNA, selected peptides and proteins and free fatty acids.
Optical flow cytometry techniques, therefore, allow individual cells to be distinguished on the basis of a large number of parameters, such as their location in the fluid stream, size, quantity of granular structures, and the presence and abundance of detectable markers. As result of this capability, optical flow cytometers are often used to generate a diagnostic profile of a population of particles in a biological sample. For example, flow cytometry has been effectively used to measure the decline or maintenance of immune cells during the course of treatment for HIV infection and to determine the presence or absence of tumor cells for prognosis and diagnosis of cancer patients.
Although optical flow cytometry is a powerful and versatile technique for identifying and characterizing components of biological samples, it has a number of significant limitations and drawbacks. First, the dynamic range of conventional optical flow cytometers with respect to the size of particles analyzed is narrow. As a result of this limitation, a number of different flow cytometers is often required for studying biological systems comprising a distribution of cells having different sizes. Second, proper operation of a flow cytometer requires that there are no clumps of cells or other debris present in the sample subjected to analysis, as these can block or deleteriously impact the laminar flow conditions the flow cell. Samples and sheath fluids, therefore, need to be carefully filtered to prevent obstruction of the flow. The necessary filtration is often troublesome to effectively carry out without affecting flow conditions in a deleterious manner. Third, conventional flow cytometers comprise complex instrumentation that requires highly trained operators for proper operation. Alignment and calibration of flow cytometers are not simple tasks, and need to be performed often to achieve accurate and well resolved optical classification. Fourth, correct operation of a flow cytometer requires the flow cell to be cleaned frequently and the tubing flushed and disinfected to prevent bio-film buildup and contamination. Cleaning and disinfecting is particularly important when using these methods to characterize samples containing microorganisms. Fifth, the sample under investigation is lost after analysis in many flow cytometer systems, and therefore, can not be subjected to additional analysis using complementary techniques. This is a significant disadvantage when analyzing samples available only in minute quantities or hard-to-get samples. Finally, the substantial cost of commercially available flow cytometers, even systems not capable of cell sorting, in addition to significant limitations in their portability, are disadvantages which have prevented widespread use of this technology outside large research and clinical institutions.
It will be appreciated from the foregoing that there is currently a need in the art for optical methods and devices for identifying and characterizing components of sample present in extremely low quantities, particularly trace components of biological samples. Optical analysis methods and devices providing a large dynamic range with respect to the size distribution, composition and physical state of particles analyzed are needed. In addition, optical analysis methods and devices are needed that are less susceptible to bio-film buildup and contamination than conventional optical flow cytometers, and which do not require cumbersome pre-filtration of samples undergoing analysis. Finally, inexpensive, simple and portable optical analysis devices are needed for analyzing biological samples, which can be operated by technicians without extensive training.